We have developed methods for mapping the structure and the location of active sites of proteins. These methods rely on measuring changes in solvent-accessibility in the presence and absence of ligands, as determined from the rates of exchange of solvent deuterons with amide NH (H/D-Ex). Although our methods are being extensively developed to assess the binding of inhibitors to enzymes, they have not yet been applied to characterizing changes in conformational stabilization induced by ligand binding to nuclear receptors. It has been demonstrated that different ligand chemotypes of PPARgamma induce distinct conformation stabilization having a unique affect the gene expression pattem observed following receptor activation. The goal of the current proposal is two-fold. First, we will integrate H/D-Ex with high throughput computational mapping and screening approaches recently developed in the laboratory of Dr. Vajda at Boston University. The computations will be used as filters to limit the number of compounds that will be screened by H/D-Ex. Second, we extend the integrated method to study the binding sites and conformational changes induced by nuclear receptor activators on vadous isoforms of peroxisome proliferator-activated receptor (PPARs). Our collaboration with the Vajda group provides us with the ability to correlate ligand binding with conformational changes that drive activation of the nuclear receptor complex. We have 4 specific aims in this proposal: 1) Extend H/D-Ex methods to the characterization of ligand binding sites of nuclear receptors, 2) Using H/D-EX, map conformational changes induced by PPAR ligands. Specifically we will determine the extent to which we can map conformational changes that are specific for the chemical class of ligand, and correlate confromational stabilization with the specific activity of the ligand, 3) Using computational solvent mapping, explore nuclear receptor active sites, and correlate computational mapping results with H/D-Ex data, and 4) Extend computational screening to nuclear receptors using multiple receptor structures and clustering.